Volume 15, Issue 5, Pages 981-988 (May 2007) Engineering Artificial Antigen-presenting Cells to Express a Diverse Array of Co- stimulatory Molecules  Megan M Suhoski, Tatiana N Golovina, Nicole A Aqui, Victoria C Tai, Angel Varela-Rohena, Michael C Milone, Richard G Carroll, James L Riley, Carl H June  Molecular Therapy  Volume 15, Issue 5, Pages 981-988 (May 2007) DOI: 10.1038/mt.sj.6300134 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Lentiviral-mediated expression of CD64 and CD32 in K562 artificial antigen-presenting cells (aAPCs). (a) Expression of CD64 and CD32 in K562 cells. The parental K562 cell line was transduced with a lentiviral vector expressing CD64 or CD32, and the cells were sorted and cultured as described in Materials and Methods. (b) Relative binding of anti-CD3 to CD32- and CD64-transduced K562 cells. CD32 and CD64 aAPCs were washed into serum-free Roswell Park Memorial Institute 1640 medium and cultured overnight at 37°C in 5% CO2. The aAPCs were loaded with 0.5 μg/ml anti-CD3 and stained with secondary antibody. The open histogram represents cells stained with secondary detection antibody only; the shaded histogram represents cells pre-loaded with anti-CD3 and then stained with secondary detection antibody. (c) Prolonged surface presentation of Fc-bound antibody on CD64-transduced K562 aAPCs. CD64 aAPCs were incubated in serum-free medium overnight at 37°C in 5% CO2, then loaded with 1 μg/ml per 1 × 106 of either murine IgG1 or murine IgG2a fluorescein isothiocyanate (FITC)–conjugated antibodies for 30 minutes at 4°C. FITC-conjugated Immuno-brite fluorospheres (Beckman Coulter, Fullerton, CA) were used to calculate the number of molecules bound to the CD64-transduced aAPCs. Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Efficient T-cell activation and proliferation using lentiviral-transduced K562 artificial antigen-presenting cells (aAPCs). (a) CD64-transduced K562 aAPCs were incubated with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) at the indicated concentrations and then washed. The antibody-loaded aAPCs were lethally irradiated and placed in culture with carboxyfluoroscein succinimidyl ester (CFSE)–labeled CD8 T cells at a 1:2 ratio of aAPCs to T cells. T cell proliferation was assessed on day 4 of culture by flow cytometry. (b) Efficiency of CD8 T cell activation and proliferation after various modes of stimulation. CD8 T cells from the peripheral blood of a normal donor were labeled with CFSE and stimulated with soluble anti-CD3 (10 μg/ml), phytohemagglutinin (PHA) (3 μg/ml), anti-CD3/CD28-conjugated beads (3:1 ratio of beads to T cells), and irradiated CD32-transduced or CD32- and CD86-transduced aAPCs (2:1 ratio of T cells to K562 aAPCs) in the presence of the indicated amount of anti-CD3, or anti-CD3 plus anti-CD28 at 0.5 μg/ml, or else as otherwise noted. On the first day of stimulation, 300 IU/ml recombinant interleukin-2 (IL-2) was added. CD8+ T cell proliferation was assessed on day 4 of stimulation by flow cytometry. Histograms represent CFSE profiles of gated CD8+ T cells. (c) T cell proliferation induced by an “off-the-shelf” aAPC. Anti-CD3-loaded, lethally irradiated fresh or cryopreserved CD64-86-transduced K562 cells mediate efficient activation and growth of CD4 T cells. K562 cells expressing CD64 and CD86 were cryopreserved with or without pre-loaded anti-CD3 (OKT3) at 1, 10, or 50 μg/ml. Pre-loaded aAPCs were thawed for immediate use, and freshly prepared OKT3-loaded aAPCs were added as indicated to CFSE-labeled CD4+ T cells (2:1 ratio of T cells to K562 aAPCs). T cells were also stimulated using anti-CD3/CD28 beads and proliferation was assessed on day 4 of culture by flow cytometry. Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Lentiviral-mediated expression of multiple genes and functional peptide presentation in K562-based artificial antigen-presenting cells (aAPCs). (a) K562 cells were transduced with lentiviral vectors containing gene inserts for human lymphocyte antigen (HLA)-A2, CD32, CD80, CD83, CD86, CD137L (4-1BBL), and CD252 (OX40L) and sorted with MOFLO to isolate single-cell clones. Clones were cultured for 2 months without antibiotics, and expression was detected by flow cytometry. Histograms represent protein expression in the CD32-80-83-86-137L-252-A2 cell line (shaded) compared with parental K562 cells (open). (b) K562 cells were transduced with lentiviral vectors for CD32 and HLA-A*0201 and sorted based on surface expression. The K562 aAPC targets (T) were pulsed overnight with peptides and labeled with 51Cr as described in Supplementary Materials and Methods. Peptide-pulsed aAPCs or anti-CD3-loaded aAPCs were cultured with a flu-specific CD8+ effector clone (E) isolated from an HLA-A2+ donor. Cultures were harvested after 4 hours, and CTL activity, measured by percentage specific lysis, was calculated for each target: flu-peptide pulsed (squares), HTLV-1 tax control peptide (diamonds), and anti-CD3-loaded (triangles). Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Formation of artificial antigen-presenting cell (aAPC)–T cell clusters as viewed with an inverted phase-contrast microscope. The relative capacity of aAPCs to aggregate T cells is shown in the presence and absence of anti-CD3 (OKT3, 0.5 μg/ml). K562 cells transduced with CD32, CD80, CD83, CD86, and CD137L (4-1BBL) were lethally irradiated and mixed with CD8 T cells at a 1:2 ratio. (a) CD32-80-83-86-137L aAPC only; (b) CD8 T cells only; (c) CD8 T cells and aAPC only; (d) CD8 T cells and anti-CD3/CD28 beads; (e) CD8 T cells and anti-CD3; (f–h) CD8 T cells, aAPC, and anti-CD3. Micrographs a–g and h depict days 2 and 3 of culture, respectively. Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Artificial antigen-presenting cells (aAPCs) mediate helper-independent expansion of CD8 T-cell subsets. Efficient induction of CD8 T-cell subset proliferation using anti-CD3-loaded CD32-80-83-86-137L aAPCs. CD8 T cells were divided into CD28+ and CD28− subsets by flow sorting. The T-cell subsets were carboxyfluoroscein succinimidyl ester (CFSE)–labeled and cultured with media and soluble anti-CD3 (OKT3, 0.5 μg/ml), anti-CD3/CD28 beads (3:1 ratio of beads to T cells), or CD32-80-83-86-137L loaded with anti-CD3 (2:1 ratio of T cells to aAPCs). Exogenous cytokines were not added to the culture. Overlay of CFSE histograms on day 8 of culture is shown. Inset dot plots represent expression of CD28 (y-axis) and CD8 (x-axis) on cells recovered on day 8 after stimulation with anti-CD3-loaded CD32-80-83-86-137L aAPC. mAb, monoclonal antibody. Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 K562 artificial antigen-presenting cell (aAPC)–expanded CD8 T cells retain cell surface phenotype and ability to produce cytokines. Bulk CD8 T cells were expanded for two rounds of stimulation with anti-CD3/CD28 beads or the indicated aAPCs loaded with anti-CD3 as described in the Materials and Methods. When the cells returned to resting state, their phenotype was analyzed using flow cytometry and compared with CD8 T cells from day 0 or cryopreserved for future characterization. (a) Cell surface expression of CD28, CD27, and CD62L was measured for each condition. Quadrants were designed based on negative isotype control, and cells were gated on live CD8+ cells. (b) Induction of cytokine production in K562 aAPC–expanded CD8 T cells. Cryopreserved CD8 T cells from day 0 or expanded with the K562 aAPCs indicated were thawed and incubated for 5 hours in medium alone (top panel) or with phorbol myristate acetate (PMA) and ionomycin (iono) (bottom panel) and assessed for intracellular interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Non-overlapping roles of CD80 and CD137L (4-1BBL) in helper-independent expansion of human CD8 T cells with artificial antigen-presenting cells (aAPCs). CD8 T cells (1 × 106) were stimulated with 0.5 × 106 irradiated aAPCs pre-loaded with anti-CD3 expressing CD32-CD137L (open squares) or CD32-80-137L (triangles), or stimulated with 3 × 106 anti-CD3/28 beads (circles), or cultured in media plus anti-CD3 (dotted line). CD8 T cells were re-stimulated when cells returned to resting volume (250 fl–300 fl) on day 8–12 in culture with re-addition of aAPCs or beads, as indicated by the arrow or arrowhead. No exogenous cytokines were added to the T-cell cultures. Figures are representative of at least three independent experiments using multiple normal donor CD8 T cells. (a) Total number of CD8 T cells in culture. (b) T-cell volume (fl) as determined by electronic cell sizing. (c) T-cell volume (fl) of CD8 T cells expanded with CD32 aAPCs expressing CD80 alone (shaded squares) or CD137L alone (open squares) or CD80 and CD137L (open triangles). Cultures were re-stimulated when they returned to resting volume, as indicated by the arrow or arrowhead. Re-stimulation of cells stimulated with anti-CD3-loaded CD32-137L aAPCs occurred on day 9 of culture (gray arrow). Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 K562 artificial antigen-presenting cell (aAPC) supports expansion of lentivirally transduced CD8 T cells. (a) Schematic of the experimental protocol. (b) Phenotype of cells assessed on day 17 in culture by flow cytometry. Histograms in top panel represent green fluorescent protein (GFP) expression. Bottom panel shows quadrants of GFP (x-axis) and CD28 (y-axis) compared with isotype control on far right. (c) Growth curve of cell counts and (d) mean cell volume graphs represent first stimulation (beads and 24-hour transduction) followed by re-stimulation indicated by the arrow with anti-CD3/CD28 beads (triangles) or anti-CD3-loaded CD32-137L aAPCs (squares), or without re-stimulation (circles). Molecular Therapy 2007 15, 981-988DOI: (10.1038/mt.sj.6300134) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions